This application outlines methods and systems to be used in isolating the zonula occludens (ZO) from chicken intestine and mouse liver as assayed by electron microscopy. The methods involve isolating ZO-rich brush border preparations (intestine) and bile canaliculus fractions (liver), and subsequently enriching for the ZO by selective solubilization of filamentous contaminants using DNase/KCl extractions, and non-junctional membranes using detergent solubilizations and treatments with other chaotropic reagents such as urea, guanidine, and other salts. Enrichment will then be carried out with sucrose gradient ultracentrifugation and two polymer phase separation. Major peptides which co-isolate with the ZO will be identified by SDS-polyacrylamide gel electrophoresis, enriched by electroelution from the gels, and used as antigen to immunize rabbits and guinea pigs. The antigens will be localized back within the structure of the ZO with the antisera by immunocytochemistry of the brush border and canaliculus fractions. Chemical cross-linking will be used to aid in the diagnosis of ZO polypeptides. The permeability of the ZO with time will be investigated with horseradish peroxidase in intestine and in a cultured kidney cell line (MDCK).